ADP is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. ADP causes platelets to change their shape, to aggregate, to release contents of granules, and to produce thromboxane A2, another potent agonist for platelets. The physiological effects and intracellular responses of ADP on platelets have been well characterized and the receptor mediating these effects has been cloned. However the molecular mechanism of these ADP-mediated physiological processes remains obscure. During the previous grant period, we have demonstrated three ADP receptor subtypes, P2Y1, P2Y12, and P2X1, on platelets and elucidated a number of signaling mechanisms in platelets. In this grant period, we propose to enhance our understanding by elucidating the molecular mechanisms of ADP-induced platelet activation. 1. We hypothesize that G protein-activated inwardly rectifying K+ channels play an important role in the Gi mediated effects on platelet fibrinogen receptor activation, platelet dense granule release, and thromboxane A2 generation. We will test this hypothesis by pharmacological approaches and by molecular genetic approaches. 2. We hypothesize that the functional P2Y12 receptor requires an environment of lipid microdomains, also known as lipid rafts. Our preliminary evidence indicates that the function of P2Y12 receptor, is selectively affected by cholesterol depletion. We will test our hypothesis and identify the basis for such requirement by biochemical and pharmacological approaches. 3. Platelets are known to express three receptors for ADP. We hypothesize that platelets express a low affinity ADP receptor that does not cause increases in intracellular calcium. We propose to provide evidence for this fourth ADP receptor on platelets by pharmacological and gene knockout approaches. 4. We hypothesize that distinct intracellular domains on P2Y1 and P2Y12 receptors couple to the Gq and Gi proteins. This hypothesis will be tested by the molecular and cell biological approaches to identify the G protein-coupling domains on the P2Y 1 and P2Y 12 receptors.